Cellobiohydrolase A (CBHA) gene cloning from Aspergillus niger to the yeast expression vector as a stages to create cellulosic ethanol strain

Abstract

Cellulosic ethanol production as alternative energy requires cellulase enzyme (endoglucanase, exoglucanase, and β-glucosidase) in the cellulose degradation process. Aspergillus niger as a recombinant DNA technology can utilize for cellulosic ethanol production. This research aims to isolate the CBHA gene from Aspergillus niger and find out the result of CBHA gene cloning into yeast expression vector with high copy number and strong promotor characteristic, here we used pWYH257. The total RNA was isolated from Aspergillus niger and made as a template to obtain cDNA by RT-PCR. The amplification of CBHA gene was carried out using the PCR and specific primer and visualized by electrophoresis. The CBHA gene inserted into pWYH257. DNA fragments cleaved by PstI and SpeI restriction enzyme. Ligation products were transformed into Escherichia coli DH10B competent cells using the electroporation method. The recombinant plasmid purified and visualized using electrophoresis. The digestion enzyme on the recombinant plasmid was used to determine the success of gene cloning. Based on this research, CBHA gene can be isolated from Aspergillus niger and show ~1,500 bp in size. The CBHA gene cloning to pWYH257 produces 7 recombinant plasmids.